20 research outputs found
High Charge Density in Peptide Dendrimers is Required to Destabilize Membranes: Insights into Endosome Evasion
Peptide dendrimers are a type of branched, symmetric,
and topologically
well-defined molecule that have already been used as delivery systems
for nucleic acid transfection. Several of the most promising sequences
showed high efficiency in many key steps of transfection, namely,
binding siRNA, entering cells, and evading the endosome. However,
small changes to the peptide dendrimers, such as in the hydrophobic
core, the amino acid chirality, or the total available charges, led
to significantly different experimental results with unclear mechanistic
insights. In this work, we built a computational model of several
of those peptide dendrimers (MH18, MH13, and MH47) and some of their
variants to study the molecular details of the structure and function
of these molecules. We performed CpHMD simulations in the aqueous
phase and in interaction with a lipid bilayer to assess how conformation
and protonation are affected by pH in different environments. We found
that while the different peptide dendrimer sequences lead to no substantial
structural differences in the aqueous phase, the total charge and,
more importantly, the total charge density are key for the capacity
of the dendrimer to interact and destabilize the membrane. These dendrimers
become highly charged when the pH changes from 7.5 to 4.5, and the
presence of a high charge density, which is decreased for MH47 that
has four fewer titratable lysines, is essential to trigger membrane
destabilization. These findings are in excellent agreement with the
experimental data and help us to understand the high efficiency of
some dendrimers and why the dendrimer MH47 is unable to complete the
transfection process. This evidence provides further understanding
of the mode of action of these peptide dendrimers and will be pivotal
for the future design of new sequences with improved transfection
capabilities
Constant-pH MD Simulations of an Oleic Acid Bilayer
Oleic acid is a simple molecule with
an aliphatic chain and a carboxylic
group whose ionization and, consequently, intermolecular interactions
are strongly dependent on the solution pH. The titration curve of
these molecules was already obtained using different experimental
methods, which have shown the lipid bilayer assemblies to be stable
between pH 7.0 and 9.0. In this work, we take advantage of our recent
implementations of periodic boundary conditions in PoissonāBoltzmann
calculations and ionic strength treatment in simulations of charged
lipid bilayers, and we studied the ionization dependent behavior of
an oleic acid bilayer using a new extension of the stochastic titration
constant-pH MD method. With this new approach, we obtained titration
curves that are in good agreement with the experimental data. Also,
we were able to estimate the slope of the titration curve from charge
fluctuations, which is an important test of thermodynamic consistency
for the sampling in a constant-pH MD method. The simulations were
performed for ionizations up to 50%, because an experimentally observed
macroscopic transition to micelles occurs above this value. As previously
seen for a binary mixture of a zwitterionic and an anionic lipid,
we were able to reproduce experimental results with simulation boxes
usually far from neutrality. This observation further supports the
idea that a charged membrane strongly influences the ion distribution
in its vicinity and that neutrality is achieved significantly far
from the bilayer surface. The good results obtained with this extension
of the stochastic titration constant-pH MD method strongly supports
its usefulness to sample the coupling between configuration and protonation
in these types of biophysical systems. This method stands now as a
powerful tool to study more realistic lipid bilayers where pH can
influence both the lipids and the solutes interacting with them
Unraveling the Conformational Determinants of Peptide Dendrimers Using Molecular Dynamics Simulations
Peptide dendrimers are synthetic
tree-like molecules composed of amino acids. There are at least two
kinds of preferential structural behaviors exhibited by these molecules,
which acquire either compact or noncompact shapes. However, the key
structural determinants of such behaviors remained, until now, unstudied.
Herein, we conduct a comprehensive investigation of the structural
determinants of peptide dendrimers by employing long molecular dynamics
simulations to characterize an extended set of third generation dendrimers.
Our results clearly show that a trade-off between electrostatic effects
and hydrogen bond formation controls structure acquisition in these
systems. Moreover, by selectively changing the dendrimers charge we
are able to manipulate the exhibited compactness. In contrast, the
length of branching residues does not seem to be a major structural
determinant. Our results are in accordance with the most recent experimental
evidence and shed some light on the key molecular level interactions
controlling structure acquisition in these systems. Thus, the results
presented constitute valuable insights that can contribute to the
development of truly tailor-made dendritic systems
Structural Effects of pH and Deacylation on Surfactant Protein C in an Organic Solvent Mixture: A Constant-pH MD Study
The
pulmonary surfactant protein C (SP-C) is a small highly hydrophobic
protein that adopts a mainly helical structure while associated with
the membrane but misfolds into a Ī²-rich metastable structure
upon deacylation, membrane dissociation, and exposure to the neutral
pH of the aqueous alveolar subphase, eventually leading to the formation
of amyloid aggregates associated with pulmonary alveolar proteinosis.
The present constant-pH MD study of the acylated and deacylated isoforms
of SP-C in a chloroform/methanol/water mixture, often used to mimic
the membrane environment, shows that the loss of the acyl groups has
a structural destabilizing effect and that the increase of pH promotes
intraprotein contacts which contribute to the loss of helical structure
in solution. These contacts result from the poor solvation of charged
groups by the solvent mixture, which exhibits a limited membrane-mimetic
character. Although a single SP-C molecule was used in the simulations,
we propose that analogous intermolecular interactions may play a role
in the early stages of the protein misfolding and aggregation in this
mixture
Protonation of DMPC in a Bilayer Environment Using a Linear Response Approximation
pH is a very important property,
influencing all important biomolecules
such as proteins, nucleic acids, and lipids. The effect of pH on proteins
has been the subject of many computational works in recent years.
However, the same has not been done for lipids, especially in their
most biologically relevant environment: the bilayer. A reason for
this is the inherent technical difficulty in dealing with this type
of periodic systems. Here, we tackle this problem by developing a
PoissonāBoltzmann-based method that takes in consideration
the periodic boundary conditions of lipid bilayer patches. We used
this approach with a linear response approximation to calculate the
p<i>K</i><sub>a</sub> value of a DMPC molecule when diluted
in zwitterionic lipids. Our results show that DMPC protonation only
becomes relevant at quite low pH values (2ā3). However, when
it happens, it has a strong impact on lipid conformations, leading
to significant heterogeneity in the membrane
Reversibility of Prion Misfolding: Insights from Constant-pH Molecular Dynamics Simulations
The prion protein (PrP) is the cause of a group of diseases
known
as transmissible spongiform encephalopathies (TSEs). CreutzfeldtāJakob
disease and bovine spongiform encephalopathy are examples of TSEs.
Although the normal form of PrP (PrP<sup>C</sup>) is monomeric and
soluble, it can misfold into a pathogenic form (PrP<sup>Sc</sup>)
that has a high content of Ī²-structure and can aggregate forming
amyloid fibrils. The mechanism of conversion of PrP<sup>C</sup> into
PrP<sup>Sc</sup> is not known but different triggers have been proposed.
It can be catalyzed by a PrP<sup>Sc</sup> sample, or it can be induced
by an external factor, such as low pH. The pH effect on the structure
of PrP was recently studied by computational methods [Campos et al. <i>J. Phys. Chem. B</i> <b>2010</b>, <i>114</i>, 12692ā12700], and an evident trend of loss of helical structure
was observed with pH decrease, together with a gain of Ī²-structures.
In particular, one simulation at pH 2 showed an evident misfolding
transition. The main goal of the present work was to study the effects
of a change in pH to 7 in several transient conformations of this
simulation, in order to draw some conclusions about the reversibility
of PrP misfolding. Although the most significant effect caused by
the change of pH to 7 was a global stabilization of the protein structure,
we could also observe that some conformational transitions induced
by pH 2 were reversible in many of our simulations, namely those started
from the early moments of the misfolding transition. This observation
is in good agreement with experiments showing that, even at pH as
low as 1.7, it is possible to revert the misfolding process [Bjorndahl
et al. <i>Biochemistry</i> <b>2011</b>, <i>50</i>, 1162ā1173]
p<i>K</i><sub>a</sub> Values of Titrable Amino Acids at the Water/Membrane Interface
Peptides
and proteins protonation equilibrium is strongly influenced
by its surrounding media. Remarkably, until now, there have been no
quantitative and systematic studies reporting the p<i>K</i><sub>a</sub> shifts in the common titrable amino acids upon lipid
membrane insertion. Here, we applied our recently developed CpHMD-L
method to calculate the p<i>K</i><sub>a</sub> values of
titrable amino acid residues incorporated in Ala-based pentapeptides
at the water/membrane interface. We observed that membrane insertion
leads to desolvation and a clear stabilization of the neutral forms,
and we quantified the increases/decreases of the p<i>K</i><sub>a</sub> values in the anionic/cationic residues along the membrane
normal. This work highlights the importance of properly modeling the
protonation equilibrium in peptides and proteins interacting with
membranes using molecular dynamics simulations
Treatment of Ionic Strength in Biomolecular Simulations of Charged Lipid Bilayers
Biological membranes are complex
systems that have recently attracted
a significant scientific interest. Due to the presence of many different
anionic lipids, these membranes are usually negatively charged and
sensitive to pH. The protonation states of lipids and the ion distribution
close to the bilayer are two of the main challenges in biomolecular
simulations of these systems. These two problems have been circumvented
by using ionized (deprotonated) anionic lipids and enough counterions
to preserve the electroneutrality. In this work, we propose a method
based on the PoissonāBoltzmann equation to estimate the counterion
and co-ion concentration close to a lipid bilayer that avoids the
need for neutrality at this microscopic level. The estimated number
of ions was tested in molecular dynamics simulations of a 25% DMPA/DMPC
lipid bilayer at different ionization levels. Our results show that
the system neutralization represents an overestimation of the number
of counterions. Consequently, the resulting lipid bilayer becomes
too ordered and practically insensitive to ionization. On the other
hand, our proposed approach is able to correctly model the ionization
dependent isothermal phase transition of the bilayer observed experimentally.
Furthermore, our approach is not too computationally expensive and
can easily be used to model diverse charged biomolecular systems in
molecular dynamics simulations
Constant-pH MD Simulations of DMPA/DMPC Lipid Bilayers
Current
constant-pH molecular dynamics (CpHMD) simulations provide
a proper treatment of pH effects on the structure and dynamics of
soluble biomolecules like peptides and proteins. However, addressing
such effects on lipid membrane assemblies has remained problematic
until now, despite the important role played by lipid ionization at
physiological pH in a plethora of biological processes. Modeling (de)Āprotonation
events in these systems requires a proper consideration of the physicochemical
features of the membrane environment, including a sound treatment
of solution ions. Here, we apply our recent CpHMD-L method to the
study of pH effects on a 25% DMPA/DMPC bilayer membrane model, closely
reproducing the correct lipid phases of this system, namely, gelāfluid
coexistence at pH 4 and a fluid phase at pH 7. A significant transition
is observed for the membrane ionization and mechanical properties
at physiological pH, providing a molecular basis for the well-established
role of phosphatidic acid (PA) as a key player in the regulation of
many cellular events. Also, as reported experimentally, we observed
pH-induced PAāPA lipid aggregation at acidic pH. By including
the titration of anionic phospholipids, the current methodology makes
possible to simulate lipid bilayers with increased realism. To the
best of our knowledge, this is the first simulation study dealing
with a continuous phospholipid bilayer with pH titration of all constituent
lipids
Molecular Details of INHāC<sub>10</sub> Binding to <i>wt</i> KatG and Its S315T Mutant
Isoniazid
(INH) is still one of the two most effective antitubercular
drugs and is included in all recommended multitherapeutic regimens.
Because of the increasing resistance of <i>Mycobacterium tuberculosis</i> to INH, mainly associated with mutations in the <i>katG</i> gene, new INH-based compounds have been proposed to circumvent this
problem. In this work, we present a detailed comparative study of
the molecular determinants of the interactions between <i>wt</i> KatG or its S315T mutant form and either INH or INH-C<sub>10</sub>, a new acylated INH derivative. MD simulations were used to explore
the conformational space of both proteins, and results indicate that
the S315T mutation did not have a significant impact on the average
size of the access tunnel in the vicinity of these residues. Our simulations
also indicate that the steric hindrance role assigned to Asp137 is
transient and that electrostatic changes can be important in understanding
the enzyme activity data of mutations in KatG. Additionally, molecular
docking studies were used to determine the preferred modes of binding
of the two substrates. Upon mutation, the apparently less favored
docking solution for reaction became the most abundant, suggesting
that S315T mutation favors less optimal binding modes. Moreover, the
aliphatic tail in INH-C<sub>10</sub> seems to bring the hydrazine
group closer to the heme, thus favoring the apparent most reactive
binding mode, regardless of the enzyme form. The ITC data is in agreement
with our interpretation of the C<sub>10</sub> alkyl chain role and
helped to rationalize the significantly lower experimental MIC value
observed for INH-C<sub>10</sub>. This compound seems to be able to
counterbalance most of the conformational restrictions introduced
by the mutation, which are thought to be responsible for the decrease
in INH activity in the mutated strain. Therefore, INH-C<sub>10</sub> appears to be a very promising lead compound for drug development